The requirement for splicing in humans is nearly ubiquitous. Human genes contain eight introns on average and greater than 93% of human genes undergo alternative splicing. These alternatively spliced forms are thought to contribute to the complexity of the human proteome. Further, alternative splicing is known to permit regulation of gene expression, such as during development and in response to environmental stimuli. Additionally, at least 15% of human diseases result from errors in splicing. Thus, to interpret the function of the human genome and to investigate human disease, we must describe qualitatively and quantitatively how the human transcriptome is spliced under a variety of conditions, the long-term goal of this project. Despite the development of genome-wide methodologies to assay alternative splicing, current methods are lacking in one respect or another. For example, while splicing-sensitive microarrays provided the first genome-wide view of splicing, such microarrays require prior knowledge of splice junctions and suffer from cross hybridization of splice junction probes with unspliced pre-mRNAs. While deep sequencing circumvents these limitations and offers tremendous promise, current applications of deep sequencing to splicing fail to exploit the full power of deep sequencing. Further, both approaches fail to reveal critical features of the splicing mechanism, often fail to report changes in splicing promptly, and in many cases fail to distinguish alternative splicing from transcriptional regulation. We propose to overcome the limitations of existing approaches by developing and validating a new, complementary and transformative method to assay splicing genome-wide. The limitations of current methods can be attributed to their nearly exclusive focus on the mRNA product of splicing. We propose to determine the feasibility of interrogating the other product of splicing - the excised intron. While this approach carries some risk in part due to the general functional irrelevance of the excised intron product, the excised intron offers the potential to utilize the full power of deep sequencing to analyze splicing quantitatively and qualitatively in a cost-effective manner. Toward developing and testing such a method, we propose to accomplish the following three specific aims. First, we aim to purify excised introns for library construction and deep sequencing. Second, we aim to develop methods for constructing sub-libraries of the transcriptome that are rich in intronic splice sites. Third, we aim to deep sequence intronic splice site libraries and to compare an analysis of this data with microarray and deep sequencing analysis of mRNA. We propose to test this methodology initially in the facile model organism budding yeast, because of its small genome size, small intron number and simple mode of splicing regulation. Additionally, budding yeast offers existing microarray and deep sequencing datasets that will permit an immediate comparative evaluation of this new method. By focusing the entire power of sequencing on splicing events reflected in excised introns, we expect to enable a new level of discovery and analysis of splicing that is currently inaccessible. PUBLIC HEALTH RELEVANCE: Translation of the information encoded in our DNA into the molecular workhorses of the cell requires an intermediate step, termed RNA splicing, in which interruptions of the information are deleted. Errors in RNA splicing account for at least 15% of all human diseases. In this project, we aim to develop a new method to analyze splicing genome-wide that will reveal unprecedented insights.